1. Field of the Invention
The present invention relates to an X-ray microscope for obtaining a transmitted X-ray microscopic image of a specimen such as biological specimen by irradiating the specimen with X-rays and exciting radiation rays.
2. Related Art Statement
Various studies and developments for X-ray radiation sources and X-ray optical elements have been advanced, and one of their application systems, an X-ray microscope has been proposed. In X-ray microscope, there are provided various imaging optical elements such as Wolter type optical element which is a kind of the grazing incident optical element, a zone plate optical system utilizing diffraction, and a Schwarzschild optical system including two spherical mirrors having multilayer coatings applied thereon. Particularly, a soft X-ray microscope using soft X-rays has been developed to a study of biological substances, because damage to the biological substances can be reduced. That is to say, in the soft X-ray microscope, the biological specimens can be observed with a high resolution without dyeing or staining. Generally, a wavelength range of the soft X-rays extends from 2.ANG. which is the longest wavelength of the hard X-rays to 1000.ANG. which is the shortest wavelength of the vacuum ultraviolet rays, so that the wavelength region of the soft X-rays partially overlaps with a wavelength region of extreme ultraviolet rays.
FIG. 1 is a schematic view showing the Wolter optical element, in which X-rays are made incident upon reflecting surfaces illustrated by solid lines at large incident angles (grazing incident) and are reflected thereby due to the total reflection. FIG. 2 is a schematic view depicting the Fresnel zone plate optical element, in which X-rays are reflected by diffraction. FIG. 3 is a schematic view showing the Schwarzschild optical element using two spherical mirrors each having a multilayer coating applied thereon. There X-ray optical elements are well-known in the art and are described in "X-RAY OPTICAL ELEMENTS and THEIR APPLICATIONS", Sadao AOKI, Applied Physics, Vol. 56, No. 3, 1987, pp. 342(44)-351(53), so that their detailed explanation is omitted here.
Among the soft X-ray wavelength region, soft X-rays within a wavelength region of .lambda.=43.7.ANG. to 23.6 .ANG., i.e. a so-called water window region between the K.alpha. absorption edge of carbon and the K.alpha. absorption edge of oxygen are important, because the absorption of the soft X-rays of this region by carbon and nitrogen is large, while that by water composed of oxygen and hydrogen is small. Therefore, by using the soft X-rays of the water window region, it is possible to observe biological specimens mainly composed of proteins (living tissues) with high resolution in water. Due to this fact, research institutions have endeavored to develop optical elements, radiation sources and detectors having high performance for the soft X-rays of the wavelength region of 43.7 to 23.6.ANG..
As stated above, the soft X-rays within the above mentioned wavelength region are suitable for inspecting the biological substances, however it is practically difficult to manufacture the optical elements, radiation sources, detectors and so on having excellent property due to the following reason. Firstly, it is very difficult to manufacture the X-ray multilayer reflecting mirror and filter having superior characteristics for the soft X-rays of the above mentioned wavelength region. That is to say, upon designing the multilayer reflecting mirror having a high reflectance, it is required that two kinds of substances with the largest possible difference between their refractive indices are built up alternately to form a multilayer film. However, the refractive indices of almost all substances for the X-rays are close to unity, and thus it is difficult to choose two kinds of substances with the large difference in the refractive index. Although a proposal has been made few materials of the multilayer coating whose reflectances are expected to be somewhat improved, such as multilayer films Ni/Sc and Ni/Ti having a structure of laminating alternately Ni (nickel) and so (scandium) and Ni and Ti (titanium), these materials are liable to be crystallized during the evaporation, and this makes it difficult to deposit a uniform film. Furthermore, when the normal incident mirrors are to be formed by the presently developed technique, a period or pitch of a multilayer coating for the wavelength region of 43.7 to 23.6.ANG. becomes smaller than 20.ANG., so that the fabrication of the thin multilayer film is difficult. Still further, in the wavelength region of 43.7 to 23.6.ANG., the absorption of X-rays in terms of carbon is high and thus it is impossible to utilize organic materials as filters and a choice of filter materials is limited. In the X-ray microscope, it is necessary to provide the multilayer coatings and filters, but the above problems become obstacles upon utilizing the soft X-rays.
Even though the above mentioned first problem were solved, there is remained a second problem which will be explained next. This second problem relates to a quality of the transmitted X-ray image of a specimen, particularly the decrease in contrast of the image. That is to say, the absorption of the soft X-rays by a living specimen is determined by a thickness of the specimen, a density of nitrogen contained in the specimen and a wavelength of the X-rays, and therefore when the specimen has a large thickness and a high density of nitrogen, a substantial part of the X-rays is absorbed by the specimen and thus the transmitted X-ray image of the specimen becomes dark. When a thin specimen having a low nitrogen density is observed, almost all incident X-rays are transmitted through the specimen and thus a transmitted X-ray image becomes bright. In both cases, the constant of the transmitted X-ray image is very low.
The above mentioned second problem could be solved by adjusting the thickness of the specimen or by adjusting the wavelength of the X-rays within the wavelength region of 43.7 to 23.6.ANG., because the nitrogen density of the specimen could never be artificially adjusted. In the first solution, the thickness of the specimen is adjusted with the aid of a precision machine such as a microtome which requires high operator skill for cutting the specimen to reduce its thickness. Further, the cutting operation has to be repeated through the rule of trial and error and requires a long time. Therefore, this solution is not practical at all. The second solution requires a wide change in design and layout of the microscope optical systems in using the X-ray optical elements such as zone plate and Schwarzschild optical element, so that this solution is also of little practical use and at variance with the reality.
There has been proposed an X-ray microscope using X-rays of a wavelength region of, e.g. 65 to 43.7.ANG. other than the above mentioned region of 43.7 to 23.6.ANG. in which a microscopic image of a specimen of a particular protein molecule can be obtained with high contrast. Now a principle of this X-ray microscope will be explained with reference to FIGS. 4 and 5. FIGS. 4A to 4F represent the transition process of electron in carbon atom upon absorbing X-rays. FIG. 4A shows an electron arrangement within the carbon atom in the ground state. When the carbon atom is irradiated with X-rays, an electron E in the 1s orbit is ionized as illustrated in FIG. 4B (this is referred to as a first transition) and a hole is formed in the 1s orbit as depicted in FIG. 4C. This condition is very unstable in the view point of energy, so that an electron in the 2p orbit is transfers into the 1s orbit (this is termed as a second transition) to secure its stability as shown in FIG. 4D. When the carbon atom constitutes a protein molecule, a hole formed in the 2p orbit (see FIG. 4E) captures an electron (a third transition) from a surrounding constituent element to resume the initial ground state as shown in FIG. 4F. During the above mentioned transition process, the transmitted X-ray microscopic image of protein is obtained by utilizing the first transition. However, if a wavelength of the used X-rays is longer than the absorption edge of the carbon, the X-rays could not be absorbed by the protein, and thus the contrast of the obtained microscopic image is decreased extremely.
Now considering the preceding electron transitions from their reverse processes, it is recognized that even though the wavelength of the X-rays is longer than the absorption edge of carbon, the transmitted X-ray microscopic image of protein can be observed with high contrast. At first, from the ground state shown in FIG. 5A, and electron E in the 2p orbit is excited or ionized due to an reversed third transition to form a hole in the 2p orbit as shown in FIG. 5B. Then, as illustrated in FIG. 5C, an electron in the 1s orbit is excited by the irradiation of X-rays into the 2p orbit due to the reversed second transition as depicted in FIG. 5D. This reversed second transition can be performed by the X-rays having a photon energy which is lower than the wavelength of the absorption edge of carbon. That is to say, the reversed second transition can be carried out by the X-rays having a wavelength longer than the absorption edge of carbon.
The condition of FIG. 5D is entirely identical with the condition of FIG. 4B which is obtained after the first transition for ionizing the electron in the inner-shell 1s from the ground state, but an energy for ionizing or exciting the electron from the 1s orbit into the 2p orbit is about several to twenty eV (corresponding to a wavelength region of 100 to 300 nm), so that the reversed second transition may be performed by means of an ultraviolet laser. An energy required for exciting the electron from the 1s orbit to the 2s orbit in FIG. 5D is smaller than an energy required for ionizing the inner-shell electron in FIG. 4B by several to 20 eV. Therefore, by using the two step transition including the process for exciting the electron in the 2p orbit and the process for exciting the electron in the 1s orbit into the 2p orbit as shown in FIGS. 5A to 5D, it is possible to the observe the transmitted image of protein even by using the X-rays of the wavelength longer than the absorption edge of carbon.
The superiority of the above mentioned method using the reversed transitions has been quantitatively confirmed by J. K. Klems in X-ray Absorption in Valence-excited Molecules as a Possible Contrast Mechanism for Chemically Sensitive Imaging and Spectroscopy, Physical Review A, Vol. 43, No. 4, February 1991, pp. 2041-2045. In this method, firstly the X-rays having a wavelength longer than the absorption edge of carbon can be used, and therefore the multilayer coating may be formed by materials such as W (tungsten) and C (carbon) which are excellent in optical constant and easy of film fabrication. Moreover, these materials have been studied for a long time and have been actually used. Secondly, a necessary energy for ionizing or exciting the electron in the 2p orbit differs for particular proteins, so that carbon atom in a specific protein can be selectively excited or ionized. Further, a value of energy for the succeeding electron transition from the 1s orbit into the 2p orbit is determined uniquely. Therefore, when X-rays having the equivalent photon energy are taken as a probe, it is possible to obtain the transmitted X-ray image of a desired protein. In this case, the contrast of this transmitted X-ray microscopic image is enhanced by more than one figure compared with the conventional method utilizing the wavelength region of 43.7.ANG. to 23.6.ANG. as shown in FIG. 6.
The above mentioned principle can be easily realized by slightly changing the existing X-ray microscope system. FIG. 7 is a schematic view showing the known X-ray microscope. The X-ray microscope comprises an X-ray source 1 for emitting X-rays having a given wavelength, a condenser lens 2 for projecting the X-rays onto a specimen 3, an objective lens 4, a filter 5 and a detector 6 which are arranged on the same optical axis. The objective lens 4 may be classified into two groups, i.e. a wave dispersion type such as zone plate or the Schwarzschild optical element and a grazing incident mirror type of collecting white light such as Wolter type optical element. When a white light source is used for the X-ray source 1 and the Wolter type objective lens 4 is provided, it is necessary to arrange a spectrometer on the optical path extending to the detector 6. The X-ray detector 6 may be formed by a microchannel plate (MCP) and an imaging element such as charge coupled device (CCD). When the white light radiation source is used, a thin film filter such as beryllium (Be) film for cutting off stray light rays having wavelengths longer than that of ultraviolet is generally arranged in the optical path. In order to avoid the absorption of the X-rays by the air, the above mentioned optical elements are all arranged within a vacuum chamber not shown. The X-ray detector 6 is connected to a signal processing circuit and an image signal produced by this circuit is supplied to a monitor to display a visible image of the specimen on the monitor.
The reversed transition method proposed by J. H. Klems has been applied to the above mentioned X-ray microscope by simply adding ultraviolet ray source 7, condenser lens 8 and ultraviolet (UV) reflection mirror 9 as illustrated in FIG. 8. It should be noted that in U.S. Pat. No. 5,216,699 issued on Jun. 1, 1993 and assigned to the same assignee to whom the present application is also assigned, there is described the X-ray microscope shown in FIG. 8. The UV reflection mirror 9 is inserted between the specimen 3 and the objective lens 4 and has a sufficiently high transmittance for the wavelength region of 65 to 43.7.ANG. and has a sufficiently high reflectance for the ultraviolet rays. Therefore, the UV reflection mirror 9 also serves as the X-ray filter for cutting off the noise, i.e. stray light rays having wavelengths longer than that of the ultraviolet, so that the X-ray filter 5 shown in FIG. 7 is dispensed with. In this X-ray microscope, when the specimen 3 is irradiated with the X-rays having the equivalent energy for effecting the reversed second transition shown in FIG. 4p as well as the ultraviolet rays emitted from the UV source 7 by means of the condenser lens 8 and UV reflection mirror 9, the electron in the 2p orbit or carbon of a specific protein can be ionized or excited and the transmitted X-ray microscopic image of the specimen 3 can be observed through the process of the transition proposed by J. H. Klems.
The X-ray microscope shown in FIG. 8 has further advantages. That is to say, the absorption coefficient of the X-rays due to a living specimen can be simply changed, so that the contrast of the transmitted X-ray image can be adjusted without changing the thickness of the specimen or the wavelength of the X-rays. The X-ray absorption coefficient of the living substance is proportional to the number of carbon atoms having the holes in the 2p orbits after the irradiation with the ultraviolet rays as shown in FIG. 5C and this number is proportional to a photon flux or an amount of the irradiated ultraviolet rays. Therefore, by adjusting a photon flux or an amount of the ultraviolet rays to be made incident upon the living specimen, the absorption coefficient of the X-rays in terms of the living specimen can be changed such that the contrast of the transmitted X-ray image becomes optimum.
Moreover, a transmitted X-ray image of the specimen without the irradiation with the ultraviolet rays is picked-up in addition to the transmitted X-ray image of the same specimen with the irradiation with the ultraviolet rays, and then a differential image of these two X-ray images is derived to remove background noise due to elements other than carbon. The thus obtained differential X-ray image has a superior contrast purely due to carbon.
FIGS. 9 and 10 are schematic diagrams showing another embodiments of the X-ray microscope disclosed in the above mentioned U.S. Pat. No. 5,216,699. The X-ray microscope shown in FIG. 9 comprises an X-ray source 11 formed by a synchrotron radiation (SOR) source, a spectrometer 12, a condenser lens 13 formed by a Fresnel zone plate, an objective lens 14 also formed by a Fresnel lens, and an X-ray detector 16 formed by MCP. These elements are arranged on the same optical axis. A specimen 14 to be inspected is placed between the condenser lens 13 and the objective lens 15. The X-ray microscope further comprises an ultraviolet laser light source 17, a condenser lens 18, a glass wedge 19 and a thin film 20 made of Be (beryllium) which is arranged at 45 degrees with respect to the optical axis. Ultraviolet rays emitted by the UV light source 17 is projected onto the specimen 14 by means of the Be thin film 20. This Be thin film 20 further serves to prevent assay rays such as ultraviolet rays from being incident upon the detector 16.
The wedge 19 is made of a material such as BK7 glass having a high absorption for the ultraviolet rays and is arranged movably with respect to an optical axis as shown by a double headed arrow, so that by adjusting a position of the wedge 19, an optical path length of the wedge through which the ultraviolet rays pass can be changed so as to adjust an amount of ultraviolet rays to be made incident upon the specimen 14. In this manner, a properly adjusted amount of the ultraviolet rays can be projected onto the specimen 14 together with the X-rays, and thus it is possible to obtain a transmitted X-ray microscopic image of the protein specimen having a high contrast compared with the known X-ray microscope.
The X-ray microscope illustrated in FIG. 10 is basically same as that shown in FIG. 9. In this microscope, there is provided a laser plasma source including Nd:YAG laser 21, an X-ray condenser lens 22 is formed by a Wolter type optical element, an objective lens 23 is formed by a Schwarzschild optical element, and a detector 24 is formed by a microchannel plate (MCP). In order to generate X-rays, a laser light beam emitted from the Nd:YAG laser 21 is made incident upon a target 25 by means of a half mirror 26 and a condenser lens 27. A part of the laser beam reflected by the half mirror 26 is transmitted through a polarizer 28 to adjust an amount of laser beam passing therethrough, and then the laser beam emanating from the polarizer is made incident upon an optically anisotropic or non-linear crystal 29 such as KDF (KH.sub.2 PO.sub.4). Then, the ultraviolet rays are converted into fourth order harmonica and the thus converted ultraviolet rays of harmonics are made incident upon an ultraviolet reflecting mirror 32 by means of reflection mirror 30, condenser lens 31 and UV. The ultraviolet rays reflected by the mirror 32 are then made incident upon a specimen 33.
The X-ray microscope shown in FIG. 10 has advantages compared with the X-ray microscope depicted in FIG. 9 that there is not provided the UV light source such as UV laser and the objective lens 23 is formed by the Schwarzschild optical element including the multilayer coatings made of W/C and having an excellent optical property. For instance, the reflectance of a multilayer film composed of 200 membranes coating for the normal incident ultraviolet having the wavelength of 45.ANG. amounts to about 30%, so that it is possible to observe a transmitted X-ray image having a high brightness.
In the X-ray microscopes illustrated in FIGS. 8 to 10, it is essentially required to irradiate the specimen with a sufficiently large amount of the ultraviolet rays in order to observe a transmitted X-ray microscopic image having a high contrast. However, in these X-ray microscopes, the UV light sources are arranged outside the vacuum chamber and the ultraviolet rays are made incident upon the specimen by means of the UV transmissive window provided in the wall of the vacuum chamber, so that a relatively large amount of the ultraviolet rays is absorbed by the air and window and thus an amount of the ultraviolet rays actually impinging upon the specimen is reduced. Therefore, the transmitted X-ray microscopic image having the high contrast could not be obtained. In order to avoid such a drawback, it is necessary to provide a large scale UV light source which can emit a very large amount of ultraviolet rays. But this solution results in high cost.
Furthermore, in the above mentioned X-ray microscopes, the ultraviolet rays are made incident upon the specimen as a diverging ultraviolet beam by means of the thin HE film serving as the ultraviolet reflecting mirror and X-ray filter. Therefore, a photon flux measured at a surface of the specimen is liable to be small, so that the electron in the 2p orbit could not be effectively ionized or excited and the contrast of the transmitted X-ray image is liable to be decreased. Moreover, a part of the ultraviolet rays is scattered within the vacuum chamber and stray rays are made incident upon the X-ray detector. This results in white noise in the transmitted X-ray microscopic image and deteriorates the image quality.
As stated above, in the X-ray microscopes shown in FIGS. 8 to 10, the X-ray absorption coefficient of the specimen can be changed by adjusting an amount of the ultraviolet rays impinging upon the specimen without changing a thickness of the specimen. However, the inventors of the present application have found that it is practically difficult to observe the transmitted X-ray microscopic image having a high contrast without adjusting a thickness of the specimen due to the existence of elements contained in a portion of the specimen which portion is free from the irradiation of the ultraviolet rays. In order to observe an optimum X-ray image having a good contrast, the inventors have found that a mutual relationship between a thickness of a specimen, a wavelength of the X-rays and a resolving power of tone has to be established quantitatively. However, no one has proposed such a relationship.
In the X-ray microscopes mentioned above, in order to obtain a transmitted X-ray microscopic image having a good contrast, it is necessary to determine a photon flux or an amount of the ultraviolet rays to be made incident upon the specimen. In other words, the inventors have found that the photon flux of the ultraviolet rays is one of important parameters for observing the X-ray image having an excellent quality. Moreover, if a suitable photon flux is determined, it will be possible to select a suitable ultraviolet light source. The selection of the ultraviolet light source such as a laser is a very important factor for designing and manufacturing actual products and puts a large influence upon cost and performance of products. However, there has not been established a theory for determining the photon flux of the ultraviolet rays to be made incident upon the specimen.
The inventors have further found experimently that the quality of the transmitted X-ray microscopic image obtained by the ultraviolet excitation type X-ray microscope depends on a time period during which the specimen is irradiated with the ultraviolet rays and a timing of the irradiation of the X-rays with respect to the irradiation of the ultraviolet rays. Also in this case, the photon flux of the ultraviolet to be made incident upon the specimen is an important factor.
In the ultraviolet excitation type X-ray microscope explained above, in order to observe various kinds of elements contained in a specimen or in order to observe the same element contained in different substances of the specimen, it is necessary to change a wavelength of the ultraviolet rays. However, in the above mentioned X-ray microscopes, a wavelength of the ultraviolet rays could not be changed or adjusted in accordance with objects to be observed.
As the soft X-ray prove, there has been proposed a secondary electron spectroscopic apparatus, in which particle beam such as electron beam, proton beam, positron beam, neutron beam, and photon beam is projected onto a specimen to emit secondary electrons and a power spectrum of the secondary electrons is detected. Recently, in a field of analyses for semiconductor surface, carbon containing organic substances, semiconductor process such as CVD, and organic electronic devices, there has been required to develop a new estimation by utilizing the electron spectroscopy for chemical analysis (DBCA) and the Augor electron spectroscopy, in which soft X-rays having wavelengths longer than several .ANG. are used as an optical probe. Particularly, there has been required to develop an analysis using soft X-rays having a wavelength longer than 5 --as the optical probe for investigating biological substances including oxygen (K absorption edge is 23.32.ANG.), nitrogen (K absorption edge is 30.99.ANG.), carbon (K absorption edge is 43.68.ANG.), phosphorus (L absorption edge is 94.ANG. and K absorption edge is 5.8.ANG.), calcium (L absorption edge is 35.ANG.), sodium (K absorption edge is 11.6.ANG.), magnesium (K absorption edge is 9.5.ANG.).
In presently available estimating apparatuses, a radiation source is formed by an X-ray tube, and thus use may be made of characteristic X-rays having a wavelength shorter than several .ANG.. Therefore, when a specimen mainly composed of carbon is to be estimated, its absorption coefficient is too small to yield a large amount of photoelectrons or Auger electrons, so that a sensitivity of analysis is liable to be low. Further, only the characteristic X-rays can be used, it is impossible to perform various analyses and elements can not be judged precisely.
In view of the above fact, there has been desired to develop a novel estimation using the soft X-rays having a wavelength longer than several .ANG.. However, in order to obtain white soft X-rays, it is necessary to provide a large scale synchrotron radiation source (SOR) which could be hardly utilized by general users.
In order to avoid the above mentioned drawbacks, there has been proposed in Japanese Patent Laid-open Publication Kokai Hei 4-140651 an electron spectroscopic analyzing apparatus using a laser plasma light source. In this analyzing apparatus, a laser beam having an intensity higher than 10.sup.12 W/cm.sup.2 is projected upon a target made of a metal under a pressure lower than 10.sup.-4 Torr and the target metal is brought into a plasma condition to emit soft X-rays having a wavelength longer than 5.ANG.. Therefore, the light source can be simply realized by means of easily available YAG laser and vacuum chamber.
The soft X-rays emitted by the above mentioned laser plasma can be advantageously dispersed widely by a toroidal grating monochrometer rather than by a constant-deviation monochrometer. Further, by providing a slit on a Rowland circle of the toroidal grating monochrometer, it is possible to derive soft X-rays having a given wavelength. The thus obtained soft X-rays are then made incident upon a specimen to emit secondary electrons. The secondary electrons are then detected by an electron analyzer arranged at a given angle with respect to the specimen and the energy of secondary electrons is analyzed. In this manner, elements constituting a surface or the specimen can be judged or determined precisely. That is to say, by analyzing the energy of electrons ionized by photons or Auger electrons, it can be determined how much electrons are emitted from what energy levels of what elements.
Further by selecting a wavelength of the soft X-rays, an amount of Auger electrons emitted from a specific element can be exclusively increased. For instance, when X-rays having a wavelength near the K absorption edge of carbon is projected onto the specimen, carbon KLL Auger electrons having the kinetic energy of about 250 eV can be predominantly observed, and thus an amount of carbon contained in a specimen surface can be analyzed.
By using the above mentioned electron spectroscopic analyzing apparatus, it is possible to observe Auger electrons emitted by various elements by selecting a wavelength of the X-rays, so that the analysis for elements can be performed with a very high sensitivity compared with the other type FSCA using the X-ray tube. Moreover, by scanning the wavelength of the X-rays and detecting amounts of emitted secondary electrons, it is possible to effect an analysis utilizing the extended X-ray absorption fine structure (EXAFS). Further, when an X-ray optical system such as inclined incident mirror is arranged behind the slit provided on the Rowland circle to produce an X-ray microbeam and a specimen stage is scanned with the X-ray microbeam, it is possible to obtain a two-dimensional image representing a distribution of an element under inspection. In this case, if the X-ray optical elements are formed by the Schwarzschild optical element or zone plate having a wavelength dependent dispersion, the monochrometer may be dispensed with.
However, when a specimen is composed of a plurality of substances and these substances have the same element, it is impossible to observe the element contained in a particular substance. For instance, a biological specimen contains various proteins and these proteins contain carbon, so that when the specimen is irradiated with the X-rays, every carbon elements contained in all the proteins omit the secondary electrons. Therefore, it is impossible to derive a distribution of the carbon element within a particular protein.